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Many bacterial toxins are important virulence factors, capable of instigating marked changes in the physiology of susceptible cells and tissues. Mechanisms of membrane attack by Vibrio toxins were examined on target cells, using cell physiological techniques, in particular, assays of haemolysis and radioisotope movement. Kanagawa haemolysin (KH; commercially available preparation of the thermostable direct haemolysin (TDH)) of V. parahaemolyticus caused lysis of human (but not horse or hagfish) erythrocytes that occurred (a) after colloid osmosis due to raised cation permeability, (b) independently of the KH:red cell ratio, and (c) with a monovalent cation selectivity series (reversed Eisenman VIII with a small K+ anomaly). The binding phase of KH was longer than the 1 - 2 minutes suggested by other workers. The KH-induced cation leak was (a) rapid in onset, (b) of a magnitude higher in the first ten minutes of treatment than subsequently, (c) of a multi-hit nature, (d) unaffected by a variety of membrane-active agents, and (e) inhibited by Zn2+, Cd2+ or mixing of toxin with dibutyl phthalate. Neuraminidase treatment of HRBC enhanced KH-induced cation influx and haemolysis, suggesting that additional receptors for TDH were unmasked by this treatment. In the presence of subhaemolytic KH, physiological levels of extracellular Ca2+ increased K+ influx by the Gardos channel, and Mg2+ (1.5 mM) decreased flux by the Na+/KV2Cr cotransporter. There were no significant changes to sodium pump activity. TDH and El Tor haemolysin (ETH) were purified from culture supernatants of V. parahaemolyticus and non-Ol V. cholerae, respectively. Highly purified TDH was derived from KH and used to confirm that the identified features of KH action were attributable to TDH. Although ETH also caused haemolysis by colloid osmosis secondary to increased cation permeability, it differed from TDH in its lability to air/vibration, relative magnitude of induced influx with respect to time, and selectivity series of induced lesion. Concentrated supernatants, from V. cholerae strains deleted of known virulence factors, caused morphological changes of Chinese hamster ovary cells, suggesting the presence of unidentified factors capable of perturbing cell physiology. Confirming its potential as an enterotoxin, KH (albeit at a high dose) increased the efflux rate constant for 86Rb+ from rabbit jejunocytes. These findings are discussed in the context of a possible pathogenic role for TDH in the gastrointestinal tract.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 126-135).

Aerolysin is a channel-forming protein toxin secreted by virulent Aeromonas species. The toxin binds to receptors on cells, is proteolytically activated, and then assembles into a heptameric oligomer, which inserts into the plasma membrane forming a functional channel, resulting in cell death. To further characterize these steps receptor identification, the effect of membrane domains on channel formation and the mode of cell death were investigated on T lymphomas. Screening of cell lysates for proaerolysin-binding proteins N-glycosidase and phosphatidylinositol specific phospholipase C treatment and/or purification of these proteins resulted in the identification of a group of glycosylphosphatidylinositol (GPI)-anchored proteins, which included contactin, Thy-1, and placental alkaline phosphatase. Liposomes were used to show that these proteins were receptors for aerolysin as those containing Thy1 or placental alkaline phosphatase in their membranes were at least 100-fold more sensitive to aerolysin than those without protein. Similarly, cells expressing GPI-anchored proteins were 10⁴-fold more sensitive to aerolysin than cells lacking them. This is likely the result of these proteins concentrating aerolysin on the cell surface and thus promoting oligomerization. The fact that these proteins can be localized to membrane domains known as rafts, which are enriched in sphingomyelin and cholesterol has the potential to affect oligomerization. To investigate this possibility erythrocytes and T lymphomas were treated with methyl-β-cyclodextrin, which destroys rafts by sequestering cholesterol. Raft disruption did not decrease the sensitivity of these cells to aerolysin. Similarly, aerolysin was no more active against liposomes containing placental alkaline phosphatase in raft domains than those in which the receptor was in non-raft domains. Thus raft domains do not promote channel formation by aerolysin. The mechanism of cell death was next investigated. At high toxin concentrations cell death was shown to proceed by necrosis, whereas at subnanomolar concentrations aerolysin triggers apoptosis. Using inactive aerolysin variants it was determined that apoptosis was not a result of binding to GPI-anchored proteins nor was it triggered by receptor clustering induced by oligomerization. Instead the formation of a small number of channels was shown to trigger apoptosis. Taken together these studies have helped to clarify the mode of action of aerolysin. / Graduate

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Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 175-193).

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser.